amaxa Nucleofector® II Manual Nucleofector® II Manual gene transfer begins here For more information and future program updates, check www.amaxa.com/ program_update Nucleofector® II Chip Card For future protocol updates, also check www.amaxa.com/ celldatabase Nucleofector® II CD-ROM Chapter 1 1. amaxa – Leading Transfection Technology 2-3 Chapter 2 2. Operating instructions 4-15 2.1 Restrictions 4 2.2 Maintenance 4 2.3 Safety instructions – please read carefully! 5 2.4 Waste disposal 7 2.5 Nucleofector® II components 7 2.6 Set-up instructions 8 2.7 Instructions for using the Nucleofector® II Device 9 2.7.1 Main menu and program execution 9 2.7.2 Selection of Nucleofector® programs by program code – “Free Program Choice” 9 2.7.3 Selection of Nucleofector® programs by name of cell type – “Cell Type List” 10 2.7.4 Selection and storage of Nucleofector® custom programs 10 2.7.5 Program execution 12 2.8 Additional Nucleofector® II settings and options 13 2.8.1 Change of standard settings 13 2.8.2 Motor 13 2.8.3 Display contrast 13 2.8.4 Beep on/off 14 2.8.5 Change of start mode 14 2.8.6 Language 14 2.8.7 Software version 14 2.8.8 Upgrading program software 14 2.8.9 Serial number 15 2.8.10 96-well mode 15 Chapter 3 3. Essentials for preparing a nucleofection® experiment 16-19 3.1 Cell culture 16 3.2 DNA quality 16 3.3 Temperature 16 3.4 Cell number per nucleofection sample 17 3.5 Important controls 17 3.6 General cell culture protocols 18 3.6.1 Trypsin treatment of adherent cells 18 3.6.2 Freezing cells 18 3.6.3 Thawing cells 19 3.7 Transient versus stable gene transfer 19 Chapter 4 4. Nucleofection® procedures 20-29 4.1 Nucleofection® of suspension cells 20 4.2 Nucleofection® of adherent cells 22 4.3 Analysis of gene transfer efficiency 25 4.3.1 Microscopic analysis of gene transfer efficiency 25 4.3.2 Antibody staining of living cells expressing a selected surface marker 26 4.3.3 Analysis of gene transfer efficiency using enzyme-linked assays 27 4.3.4 Analysis of gene transfer efficiency using flow cytometry 28 Chapter 5 5. Troubleshooting 30-31 Chapter 6 6. Nucleofector® error codes 32-33 Chapter 7 7. Genetic reporter systems 34-36 Appendix page 1 www.amaxa.com Contents 1. amaxa – Leading Transfection Technology Since its introduction in 2001, a constantly growing interest in the scientific community and industrial markets established amaxa’s Nucleofector® technology as a leading transfection technology all around the world. A continuously increasing number of publications from many different research areas reflect how the Nucleofector technology drives research in numerous different cell types and applications. The Nucleofector technology offers you more opportunities than any other transfection method to approach your research topics from many different angles. Transfection of any cell: With over 100 protocols for cell lines optimized by amaxa’s R&D team, more than 400 entries in the online cell data base and almost 50 Nucleofector Kits for different primary cells, the Nucleofector technology is clearly the transfection method- of-choice for any difficult-to-transfect cell type. Transfection of any substrate: The Nucleofector technology offers high flexibility with respect to applications, since the same transfection parameters apply for any nucleic acid substrate used. DNA vectors, e.g. expression plasmids or shRNA vectors, and RNA, e.g. siRNA duplexes, can be transfected using our transfection protocols. It is the ideal tool to help you answer the scientific questions in your day-to-day applications such as over- expression studies, gene silencing approaches, protein expression, generation of stable clones and many more. Constant innovation: At amaxa, we believe that listening to our users’ needs is the best way to develop innovative products that exactly meet your demands. In line with this dedication, the Nucleofector II Device was developed which offers a larger graphic display, a motor-driven carousel, the possibility to store individual programs for optimized cell lines and to perform bacterial transformations. In addition the Nucleofector II Device serves as the program delivery unit for amaxa’s latest innovation – the Nucleofector® 96-well Shuttle™ System, the first 96-well transfection unit that allows e.g. high throughput transfection of siRNA and cDNA libraries in virtually any cell, including primary cells. What has been our mission in the past will continue to be so in the future - providing you with innovative transfection solutions to help you thrive and excel in your research. page 2 www.amaxa.com 1. amaxa – Leading Transfection Technology The Nucleofector® technology The Nucleofector technology is based on two unique com- ponents, the Nucleofector Device that delivers the specifi- cally developed electrical parameters and Nucleofector Kits that contain cell-specific and optimized Nucleofector Solutions. As part of the Nucleofector technology, amaxa provides cell-type specific Optimized Protocols for many different cell lines and primary cells. An up-to-date database of the continuously growing range of Optimized Protocols can be found on our homepage at www.amaxa.com/celldatabase. If no Optimized Protocol for the cell line of interest is available, we offer a Cell Line Optimization Nucleofector Kit enabling you to easily establish the Nucleofector technology for your specific cell line. For fine-tuning after the optimization process, please contact our Scientific Support Teams: phone Europe/World: +49 (0)221-99199-400 USA: 240-632-9110 e-mail Europe/World: [email protected] USA: [email protected] page 3 www.amaxa.com 1. amaxa – Leading Transfection Technology 2. Operating instructions 2.1 Restrictions Medical use restrictions The Nucleofector® technology is intended for research and investigational use by professionals only. Please note that amaxa’s Nucleofector technology is not intended to be used for diagnostic purposes, for testing or treatment in humans. License statement amaxa GmbH is holder of various patents, patent applica- tions, copyrights and technical and scientific experience with respect to the Nucleofector® technology. Use of amaxa's Nucleofector technology and/or related software requires a license from amaxa GmbH. Purchasers are granted a non-exclusive, non-transferable license for a limited use of amaxa's Nucleofector technology and related software for research and development purposes, the terms of which are disclosed in detail in the license agreement accompanying the shipped Nucleofector Device. Commercial application is allowed under amaxa's license for for-profit-entities. Both licenses exclude in particular any right to manufacture, copy, reproduce, transmit, distribute, seIl, lease, transfer or sublicense amaxa's Nucleofector technology and/or related software to any third party. For license information contact amaxa GmbH by phone +49 (0)221-99199-0 or e-mail [email protected]. 2.2 Maintenance The Nucleofector II requires very little maintenance to assure reliable operation. To clean and disinfect the case, first unplug the power supply. Use a damp cloth to wipe down the outer case (water or 70 – 80% ethanol). Avoid wetting the cuvette holder within the cuvette carousel and the connectors located on the rear of the device. The Nucleofector II has been designed for use under a sterile hood with or without UV radiation source. Prolonged page 4 www.amaxa.com 2. Operating instructions exposure of the outer casing to UV light will lead to discolo- ration with no functional impairment of the Nucleofector. When laminar flows are sterilized on a regular basis by UV radiation overnight we recommend protection of the device by appropriate shielding or removal during extended UV exposure. The Nucleofector II is protected by two main fuses. Both are inside a receptacle incorporated in the inner power socket (see Fig. 2, page 7). In case of a blown fuse, you can easily replace it. Disconnect the Nucleofector II from power and insert a small flat screwdriver into the slot on the right-hand side of the receptacle to pull it open. To function, both the upper and lower part of the receptable must hold working fuses in the inner positions. Blown fuses can usually be identified by molten interrupted wires inside the glas tube. Only use T630mA or L250V fuses to substitute blown fuses. 2.3 Safety instructions – please read carefully! This symbol means that there is a risk of electric shock. An electric shock could cause death or personal injury. The Nucleofector II Device has been certified by internatio- nal safety standards and is safe to use when operated in accordance with this manual. This device is designed to deliver variable high voltage electrical impulses for the purpose of introducing nucleic acids into eukaryotic and prokaryotic cells. These electrical impulses can be DEADLY! Therefore, use this device with care and take the following PRECAUTIONS: › Only use the device once you have read and understood the Nucleofector II manual. The manual should be accessible for all users. Make sure that each potential user reads and understands it. › Do NOT open the device. The device does not contain user-serviceable parts. Under NO circumstances should circuit components be interfered with, as they can deliver an electric shock even when system is not in operation. page 5 www.amaxa.com 2. Operating instructions › Do NOT alter the device in any manner. › Do not obstruct the movement of the carousel of the Nucleofector. › Do NOT plug any non-amaxa-certified connectors into the external electrode sockets at the rear side of the Nucleofector device. The sockets are high-voltage outputs that can deliver electric shocks via any non-amaxa device that is connected to the Nucleofector. They are designed to connect amaxa certified add-on devices to the Nucleofector II, e.g.